The present invention relates to a method for forming a hybrid comprising a polynucleotide compound and a target polynucleotide together with preferred polynucleotide compounds and their synthesis. The polynucleotide compound can be used as a probe or as a drug.
Hybridization with A polynucleotide probe is a well known method for verifying the presence of a target polynucleotide. Hybridization is based on complementary base-pairing. When single-stranded polynucleotide probes are incubated in solution with single-stranded target polynucleotides, complementary base sequences pair to form double-stranded hybrid molecules. The double-stranded hybrid molecules can be separated from the single-stranded polynucleotide probes by chemical or physical means. See M. Grunstein and J. Wallis, METHODS IN ENZYMOLOGY, volume 68, R. W. U (Ed) (1979) pp. 379-469; A. R., Dunn, and J., Sambrook, METHODS IN ENZYMOLOGY, volume 65; part 1, (1980) pp. 468-478; Modified Nucleotides And Methods Of Preparing And Using The Same by D. C. Ward, A. A. Waldrop and P. R. Langer, European Patent Publication Number 0,063,879 published Nov. 3, 1982; DNA Probes for Infectious Disease by A. J. Berry and J. B. Peter, Diagnostic Medicine (March, 1984) pp. 1-8; and Recombinant DNA Technology: Some Applications In Clinical Microbiology by Wie-Shing Lee and James L. Bennington, Laboratory Management (April, 1985) pp. 21-26.
The probes generally comprise a polynucleotide portion and a signalling moiety portion attached to the polynucleotide. The polynucleotide portion of the probe has the ability to base-pair, i.e. hybridize to a sequence of interest or target polynucleotide. The signalling moiety portion of the probe has or produces the means by which the presence of a hybridized polynucleotide probe can be verified. the means can be, for example, fluorescence, phosphorescence, chromagen, radioactivity, or electron density.
The method of detecting a target polynucleotide utilizing a polynucleotide probe is generally carried out, for example, by first isolating a double-stranded polynucleotide comprising a target sequence therein from a sample. The double-stranded polynucleotide can be cut into smaller segments by means of restriction endonuclease digestion and the segments separated by gel electrophoresis, after which they are transferred from the gel onto a support for example, a nitrocellulose paper. Alternatively, the double-stranded polynucleotide can be fixed directly on the nitrocellulose without any prior enzyme digestion. The fixed polynucleotides are contacted with a solution containing the polynucleotide probe and the support is heated to about 80-90° C. to denature the polynucleotide double-strands. (The double-strands can also be denatured by means of alkali). The sample which now contains both the target polynucleotide and the polynucleotide probe is allowed to cool to an appropriate temperature during which time hybridization between the polynucleotide probe and the target polynucleotide takes place. After sufficient time has elapsed for hybridization to be complete, which can be for ten minutes to several hours, the fixed target polynucleotide is washed to remove all unbound polynucleotide probes. The signalling moiety portion of the polynucleotide probe is now detected, either directly, for example, by means of radioactivity or fluorescence, or indirectly, for example, by means of a chromogen formed by an enzymatic reaction.
A drawback of this method is that it requires several steps before the presence of the target polynucleotide can be verified. Namely, it requires the fixation of the target polynucleotide to a support, the contacting of the target polynucleotide with a polynucleotide probe, and the removal of all unhybridized polynucleotide probes from the support.
Recently, a method for detecting the presence of a target polynucleotide by means of a homogeneous (or one-step) nucleic acid hybridization assay was reported. The method comprises hybridizing first and second single-stranded polynucleotides, both of which contain light-sensitive labels, with a complementary single-stranded polynucleotide target from a sample such that non-radiative energy transfer occurs between the light-sensitive labels of the first and, second polynucleotides. At least one of the light-sensitive labels is of the absorber/emitter type such that energy absorbed from the other light-sensitive label is reemitted at a different wavelength. These secondary emissions can only occur if hybridization of both the first and second single-stranded polynucleotides to the target polynucleotide has taken place. The quantity of the target polynucleotides in the sample is related to the amount of secondary light emitted. See European Patent Publication No. 0,070,685 by Michael James Heller, published Jan. 26, 1983.
A drawback of this method is that it requires two separate polynucleotide probes to detect the presence of a target polynucleotide. In addition, the method requires the presence of a chemiluminescent catalyst, an absorber/emitter moiety, and chemiluminescent reagents effective for causing light emission in the presence of the chemiluminescent catalyst. Furthermore, only one label can be attached per polynucleotide probe because the light-sensitive label is attached to the sugar moiety of a terminal nucleoside. Also, the bulky labels may prevent hybridization of the complementary bases adjacent to the labels.
A method has been reported recently whereby a target polynucleotide is hybridized to a probe polynucleotide, and the resulting hybrid immobilized by its binding to an immobilized or immobilizable form of an antibody reagent selective for binding such hybrids. One embodiment is the use of an antibody selective for intercalated duplexes. This method, however, is not a homogeneous assay. In addition, the intercalating agent does not provide the signal directly upon hybridization of the probe to the target. Furthermore, the intercalating agent is attached directly to a base without a linker arm at positions required for base-pairing. See European Patent Application number 146,039 by J. P. Albarella et. al., published Jun. 26, 1985.
Fluorescent intercalating moieties attached to a polynucleotide have been reported in the literature. They are prepared by reacting the adenine or cytosine bases with a bifunctional reagent such as chloracetaldehyde to produce an aromatic tricyclic compound. See “Fluorescent Adenosine and Cytidine Derivatives” by J. R. Barrio, J. A. Secrist III, and N. J. Leonard, (1972), B.B.R.S. 46, (2), pp. 597-604, and “Physical Studies of Choloroacetaldehyde labelled fluorescent DNA” by C. H. Lee and J. G. Wetmur, (1973), B.B.R.S. 50 (3), pp. 879-85. This method has a drawback in that the bases converted to fluorescent moieties cannot base-pair, and thus such fluorescent moieties destabilize the hybridization process.